I am trying to amplify two genes, each ~2kb in length. I am looking for ways to do this at the stages of cDNA synthesis and PCR amplification. Primer design and thermocycle specifications do not seem to be the problem.
Basem M. Ahmed
- You can use general annealing temperature 55C for the first five cycles then go to your specific annealing temperature.
- increase annealing time to 2 minutes/ cycle
- increase the amount of added DNTPs from 2-6X
- 2kb is not really long sequence and ordinary taq can fulfil it but if your template is complex you can try long-amp enzyme of NEB with its recommended protocol.
- all these experimentation should be performed on known template (control) with proper controls included (negative control & no-template control.
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